Assessing the Th1/Th2 Landscape in Bladder Cancer

To evaluate the predisposition for a Th1/Th2 tumor microenvironment prior to BCG, the density of Th1 and Th2 cells in tumor-infiltrating immune cells was measured on formalin-fixed, paraffin-embedded tissue sections of bladder cancer (in the lamina propria without invasion, at the invasive front, within the neoplastic urothelium and within the papillary stroma) by immunohistochemistry, using a T-bet antibody (monoclonal rabbit antihuman T-bet, MRQ-46, prediluted, Roche) and a GATA3 antibody (monoclonal mouse antihuman GATA3, L50-823, prediluted, Roche), which we had already validated in a recently work [30 (link)]. IHC staining was performed using an automated immunostainer (BenchMark ULTRA, Ventana Medical Systems, Tucson, US) according to the manufacturer’s protocol. We manually counted the total density of positive cells for each subset in up to 5 high-power fields (HPF) in each region, using the same field of view in consecutive slides. Microscope images were taken with an Olympus BX50 microscope (40x magnification) equipped with the ProgResC10plus camera (Jenoptik, Jena, Germany). IHC evaluation was performed by an experienced uropathologist.

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Pichler R., Gruenbacher G., Culig Z., Brunner A., Fuchs D., Fritz J., Gander H., Rahm A, & Thurnher M. (2016). Intratumoral Th2 predisposition combines with an increased Th1 functional phenotype in clinical response to intravesical BCG in bladder cancer. Cancer Immunology, Immunotherapy, 66(4), 427-440.

Publication 2016

BenchMark ULTRA BX50 microscope ProgResC10plus camera

Antibody Cancer of bladder Embedded paraffin Formalin Gata3 Immunohistochemistry Lamina propria Microscope Mouse Neoplastic Predisposition Rabbit Th2 cells Tumor microenvironment Urothelium

Corresponding Organization : Universität Innsbruck

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Variable analysis

independent variables

Density of Th1 cells in tumor-infiltrating immune cells
Density of Th2 cells in tumor-infiltrating immune cells

dependent variables

Predisposition for a Th1/Th2 tumor microenvironment prior to BCG

control variables

Formalin-fixed, paraffin-embedded tissue sections of bladder cancer
Regions of analysis (lamina propria without invasion, invasive front, neoplastic urothelium, papillary stroma)
Immunohistochemistry (IHC) staining protocol using T-bet and GATA3 antibodies
Microscope and imaging equipment used
Evaluation by an experienced uropathologist

positive controls

Previously validated use of T-bet and GATA3 antibodies in a recent work [30]

negative controls

Not explicitly mentioned

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